Enhanced throughput mineral coatings for optimization of stem cell behavior

ABSTRACT

Disclosed are methods for cell transfection and regulating cellular behavior. More particularly, the present disclosure relates to methods of non-viral cell transfection and regulating cellular behavior using mineral coatings that allow for the enhanced transfection of cells. The mineral coatings bind polynucleotides and provide a source of calcium and phosphate ions to enhance transfection. The present disclosure also provides a high throughput platform for screening non-viral transfection of cells. The methods of the present disclosure also provide an advantageous polynucleotide delivery platform because the mineral coatings may be deposited on various medical device materials after being specifically developed using the high throughput screening platform.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under AR052893 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND OF THE DISCLOSURE

The present disclosure relates generally to methods for celltransfection and regulating cellular behavior. More particularly, thepresent disclosure relates to methods of using biodegradable mineralcoatings for non-viral cell transfection and regulating cellularbehavior.

Transfection is the process of introducing nucleic acids into cells.Various transfection strategies are available that generally involveopening transient pores in the cell membrane to allow the uptake ofmaterial by the cell. Two broad categories of transfection includechemical-based transfection methods and non-chemical transfectionmethods. Chemical-based transfection methods include, for example,calcium phosphate-based transfection and liposome-based transfection. Incalcium phosphate-based transfection, a buffer containing phosphate ionsis combined with a calcium chloride solution containing the DNA to betransfected to form calcium-phosphate precipitates that bind DNA. Asuspension of the precipitate is added to cells, which take up theprecipitate and DNA. In liposome-based transfection, DNA is incorporatedinto liposomes that fuse with the cell membrane to release the DNA intothe cells.

The deposition of calcium phosphate (CaP)-based materials on varioussubstrates has been used to develop bioactive interfaces for studyingits interaction with bone-forming cells. In addition, CaP mineralsubstrates bind and release biological molecules (e.g., DNA). Studieshave also been conducted to investigate the role of CaP mineralproperties (physical and chemical) in regulating cellular behavior,including stem cell attachment, proliferation, and differentiation.However, these previous studies have not been capable of systematicallystudying CaP mineral effects on stem cell proliferation anddifferentiation. Taken together, the potential impact of these materialson non-viral transfection and stem cell behavior as well as the inherentcomplexity of CaP requires novel strategies to identify useful CaPmineral coatings. Accordingly, there exists a need to develop non-viraltransfection methods and systems for use with these methods.

SUMMARY OF THE DISCLOSURE

The present disclosure relates generally to methods for celltransfection and regulating cellular behavior. More particularly, thepresent disclosure relates to methods of non-viral cell transfection andregulating cellular behavior using mineral coatings. The methods ofnon-viral transfection allow for the enhanced transfection of cells. Themineral coatings bind polynucleotides and provide a source of calciumand phosphate ions for transfection of cells. The present disclosurealso provides a high throughput platform for screening non-viraltransfection of cells. The methods of the present disclosure alsoprovide an advantageous polynucleotide delivery platform because themineral coatings may be deposited on various medical device materialsafter the mineral coating has been specifically developed using the highthroughput screening platform to determine the appropriate mineralcoating for the transfection of a particular cell in a particularenvironment or condition.

In one aspect, the present disclosure is directed to a method ofnon-viral transfection. The method includes preparing a substratecomprising a mineral coating, wherein the mineral coating is formed byincubating the substrate in a simulated body fluid. The simulated bodyfluid comprises from about 5 mM to about 12.5 mM calcium ions, fromabout 2 mM to about 12.5 mM phosphate ions, from about 4 mM to about 100mM carbonate ions, and a pH of from about 5 to about 7.5. Apolynucleotide is then contacted with the substrate comprising themineral coating, wherein the polynucleotide binds to the mineralcoating. Then a cell is contacted with the mineral coating; and the cellis cultured.

In another aspect, the present disclosure is directed to a highthroughput non-viral transfection system comprising a substratecomprising: a plurality of mineral coatings, wherein the plurality ofmineral coatings comprises a calcium to phosphate ratio of from about2.5:1 to about 1:1; a polynucleotide bound to the plurality of mineralcoatings; and a plurality of cells.

In another aspect, the present disclosure is directed to a method ofscreening non-viral transfection of cells comprising: culturing aplurality of cells on a plurality of mineral coatings comprising apolynucleotide bound to the mineral coatings, wherein the mineralcoatings are formed by hydrolyzing poly(α-hydroxy ester) coatings on asubstrate comprising a plurality of wells; incubating the hydrolyzedpoly(α-hydroxy ester) coatings in a plurality of simulated body fluidscomprising a calcium ion concentration of from about 5 mM to about 12.5mM, a phosphate ion concentration of from about 2 mM to about 12.5 mM, acarbonate ion concentration of from about 4 mM to about 100 mM, and a pHof from about 4 to about 7.5.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure will be better understood, and features, aspects andadvantages other than those set forth above will become apparent whenconsideration is given to the following detailed description thereof.Such detailed description makes reference to the following drawings,wherein:

FIGS. 1A-1F are scanning electron micrographs of calcium-phosphatemineral coatings grown on poly lactide glycolide (PLG) films in 2×simulated body fluid (SBF), 3.5×SBF and 5×SBF as discussed in Example 2.

FIG. 2 shows an X-ray diffraction spectra of commercially availablehydroxyapatite and calcium-phosphate mineral coatings grown on polylactide glycolide (PLG) films in 2×SBF, 3.5×SBF and 5×SBF as discussedin Example 2.

FIG. 3 shows a Fourier transform infrared (FTIR) spectra of commerciallyavailable hydroxyapatite and calcium-phosphate mineral coatings grown onpoly lactide glycolide (PLG) films in 2×SBF, 3.5×SBF and 5×SBF asdiscussed in Example 2.

FIG. 4 is a graph showing the change of calcium amount aftercalcium-phosphate mineral coatings grown on PLG films in 2×SBF, 3.5×SBFand 5×SBF were immersed in solutions containing 0×, 0.3×, 0.6× or 1×physiological calcium and phosphate concentrations at pH 7.4 asdiscussed in Example 3.

FIG. 5 is a graph showing the cumulative change of calcium amount overtime after calcium-phosphate mineral coatings grown on PLG films in2×SBF, 3.5×SBF and 5×SBF were immersed in solutions containing 0×, 0.3×,0.6× or 1× physiological calcium and phosphate concentrations at pH 7.4as discussed in Example 3.

FIG. 6 is a graph showing the change of calcium amount aftercalcium-phosphate mineral coatings grown on PLG films in 2×SBF, 3.5×SBFand 5×SBF were immersed in solutions containing 1× physiological calciumand phosphate concentrations at varying pH as discussed in Example 3.

FIG. 7 is a graph showing the cumulative change of calcium amount overtime after calcium-phosphate mineral coatings grown on PLG films in2×SBF, 3.5×SBF and 5×SBF were immersed in solutions containing 1×physiological calcium and phosphate concentrations at varying pH asdiscussed in Example 3.

FIG. 8 is a graph showing the cumulative DNA released over time aftercalcium-phosphate mineral coatings grown on PLG films in 2×SBF, 3.5×SBFand 5×SBF were immersed in solutions containing 0×, 0.3×, 0.6× or 1×physiological calcium and phosphate concentrations at pH 7.4 asdiscussed in Example 4.

FIG. 9 is a graph showing the cumulative DNA released aftercalcium-phosphate mineral coatings grown on PLG films in 2×SBF, 3.5×SBFand 5×SBF were immersed in solutions containing 1× physiological calciumand phosphate concentrations at varying pH as discussed in Example 4.

FIG. 10 is a schematic illustration showing the effect of mineralcoating morphology on cell density as discussed in Example 5.

FIG. 11A is a graph showing C₃H₁₀T½ cell proliferation on mineralcoatings as a function of time as discussed in Example 5.

FIG. 11B is a graph showing hMSC proliferation on mineral coatings as afunction of time as discussed in Example 5.

FIG. 12 is a graph showing hMSC proliferation on mineral coatings in MEMand MEM+OS at day 8 as discussed in Example 5.

FIG. 13 is a graph showing the calcium/phosphate ratio of mineralcoatings grown on PLG films in 2×SBF, 3.5×SBF and 5×SBF having varyingcarbonate concentrations as discussed in Example 6.

FIG. 14 is a graph showing the crystallinity index of mineral coatingsgrown on PLG films in 2×SBF, 3.5×SBF and 5×SBF having varying carbonateconcentrations compared to hydroxyapatite as discussed in Example 6.

FIG. 15 is a graph showing the crystallinity effect on mineral stabilityof mineral coatings grown on PLG films in 2×SBF, 3.5×SBF and 5×SBFhaving varying carbonate concentrations as discussed in Example 6.

FIGS. 16A-16C are graphs showing the dissolution of mineral coatings inDMEME serum-free medium as discussed in Example 6.

FIG. 17 is a graph showing pDNA complex binding efficiency afteradsorption and co-precipitation on mineral coatings grown on PLG filmsin 2×SBF, 3.5×SBF and 5×SBF having varying carbonate concentrations asdiscussed in Example 7.

FIG. 18A is a graph showing the luciferase activity of C₃H₁₀T½ cellscultured on mineral coatings grown on PLG films in 2×SBF, 3.5×SBF and5×SBF having varying carbonate concentrations as discussed in Example 7.

FIG. 18B is a graph showing the luciferase activity of hMSC cultured onmineral coatings grown on PLG films in 2×SBF, 3.5×SBF and 5×SBF havingvarying carbonate concentrations as discussed in Example 7.

FIG. 19 is a graph showing the luciferase activity of HUVECs cultured onmineral coatings grown on PLB films having varying mineral propertiesand pDNA complex amounts as discussed in Example 8.

FIGS. 20A-20D are photographic images (A, B) and SEM images (C, D)showing mineral coating formation on 3-dimensional PLG scaffolds beforemineralization (A, C) and after mineralization (B, D) as discussed inExample 9. Scale bars, 1 mm for photograph images and 100 nm for SEMimages.

FIG. 21 is a graph showing luciferase activity of hMSCs after 2 daysculture on mineralized 3-dimensional PLG scaffolds containing pDNAcomplexes as discussed in Example 9. Data represents mean±SD (n=4).

While the disclosure is susceptible to various modifications andalternative forms, specific embodiments thereof have been shown by wayof example in the drawings and are herein described below in detail. Itshould be understood, however, that the description of specificembodiments is not intended to limit the disclosure to cover allmodifications, equivalents and alternatives falling within the spiritand scope of the disclosure as defined by the appended claims.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure belongs. Although any methods andmaterials similar to or equivalent to those described herein may be usedin the practice or testing of the present disclosure, the preferredmaterials and methods are described below.

In accordance with the present disclosure, methods have been developedfor non-viral transfection of cells. Surprisingly, use of the mineralcoatings of the present disclosure for transfection showed 150-foldgreater transfection rates when compared to current transfectionmethods. The mineral coatings bind polynucleotides and provide a sourceof calcium and phosphate ions to enhance transfection. The presentdisclosure also provides a high throughput platform for screeningnon-viral transfection of cells. The methods of the present disclosurealso provide an advantageous polynucleotide delivery platform becausethe mineral coatings may be deposited on various medical devicematerials after being specifically developed using the high throughputscreening platform.

Methods of Non-Viral Transfection

In one aspect, the present disclosure is generally directed to a methodof non-viral transfection by forming a mineral coating on a substrate,wherein the mineral coating is formed by incubating the substrate in asimulated body fluid (SBF); contacting the substrate with apolynucleotide, wherein the polynucleotide binds to the mineral coating;contacting a cell with the mineral coating; and culturing the cell.

Suitable substrates on which the mineral coating may be includepolymers, ceramics, metals, glass and combinations thereof in the form,for example, of particles, films, dishes, plates, coverslips and slides.Particularly suitable particles may be, for example, agarose beads,latex beads, magnetic beads, and combinations thereof. Particularlysuitable dishes may be, for example, culture dishes. Particularlysuitable plates may be, for example, microtiter plates having, forexample, 4, 6, 14, 96, or more sample wells. Suitable substrates may bepolymers, ceramics, metals, glass and combinations thereof.

The substrates may be initially coated with a poly(α-hydroxy ester)film, for example. Particularly suitable poly(α-hydroxy esters) may be,for example, poly(L-lactide), poly(lactide-co-glycolide),polys-caprolactone), and combinations thereof. It should be understoodthat when making any combinations of the above films, the films aretypically mixed in suitable organic solvents as known in the art.Further, differences in molecular weights, crystallization rates, glasstransition temperatures, viscosities, and the like should be taken intoconsideration as well as understood in the art to prevent phaseseparation and lack of uniformity in the final substrates. Phaseseparation and lack of uniformity can further be avoided by altering themixing ratio of the films used in the substrate.

After preparing a poly(α-hydroxy ester) film on the substrate, thesurface of the film coating is hydrolyzed under alkaline conditions tocreate a surface having COOH and OH groups. After surface hydrolyzing,the substrate is incubated in a simulated body fluid containing asuitable mineral-forming material to form a mineral coating. Suitablemineral-forming materials may be, for example, calcium, phosphate,carbonate, and combinations thereof.

The simulated body fluid (SBF) for use in the methods of the presentdisclosure typically includes from about 5 mM to about 12.5 mM calciumions, including from about 7 mM to about 10 mM calcium ions, andincluding about 8.75 mM calcium ions; from about 2 mM to about 12.5 mMphosphate ions, including from about 2.5 mM to about 7 mM phosphateions, and including from about 3.5 mM to about 5 mM phosphate ions; andfrom about 4 mM to about 100 mM carbonate ions.

In some embodiments, the SBF may further include about 145 mM sodiumions, from about 6 mM to about 9 mM potassium ions, about 1.5 mMmagnesium ions, from about 150 mM to about 175 mM chloride ions, about 4mM HCO₃ ⁻, and about 0.5 mM SO₄ ²⁻ ions.

The pH of the SBF may typically range from about 4 to about 7.5,including from about 5.3 to about 6.8, including from about 5.7 to about6.2, and including from about 5.8 to about 6.1.

Suitable SBF may include, for example: about 145 mM sodium ions, about 6mM to about 9 mM potassium ions, about 5 mM to about 12.5 mM calciumions, about 1.5 mM magnesium ions, about 150 mM to about 175 mM chlorideions, about 4.2 mM HCO₃ ⁻, about 2 mM to about 5 mM HPO₄ ²⁻ ions, andabout 0.5 mM SO₄ ²⁻ ions. The pH of the simulated body fluid may be fromabout 5.3 to about 7.5, including from about 6 to about 6.8.

In one embodiment, the SBF may include, for example: about 145 mM sodiumions, about 6 mM to about 17 mM potassium ions, about 5 mM to about 12.5mM calcium ions, about 1.5 mM magnesium ions, about 150 mM to about 175mM chloride ions, about 4.2 mM to about 100 mM HCO₃ ⁻, about 2 mM toabout 12.5 mM phosphate ions, and about 0.5 mM SO₄ ²⁻ ions. The pH ofthe simulated body fluid may be from about 5.3 to about 7.5, includingfrom about 5.3 to about 6.8.

In another embodiment, the SBF includes: about 145 mM sodium ions, about6 mM to about 9 mM potassium ions, from about 5 mM to about 12.5 mMcalcium ions, about 1.5 mM magnesium ions, about 60 mM to about 175 mMchloride ions, about 4.2 mM to about 100 mM HCO₃ ⁻, about 2 mM to about5 phosphate ions, about 0.5 mM SO₄ ²⁻ ions, and a pH of from about 5.8to about 6.8, including from about 6.2 to about 6.8.

In yet another embodiment, the SBF includes: about 145 mM sodium ions,about 9 mM potassium ions, about 12.5 mM calcium ions, about 1.5 mMmagnesium ions, about 172 mM chloride ions, about 4.2 mM HCO₃ ⁻, about 5mM to about 12.5 mM phosphate ions, about 0.5 mM SO₄ ²⁻ ions, from about4 mM to about 100 mM CO₃ ²⁻, and a pH of from about 5.3 to about 6.0.

The mineral coatings may be formed by incubating the substrate with theSBF at a temperature of about 37° C. for a period of time of from about3 days to about 10 days.

After completion of the mineral coating preparation, the mineralcoatings may be analyzed to determine the morphology and composition ofthe mineral coatings. The composition of the mineral coatings may beanalyzed by energy dispersive X-ray spectroscopy, Fourier transforminfrared spectrometry, X-ray diffractometry, and combinations thereof.Suitable X-ray diffractometry peaks may be, for example, at 26° and 31°,which correspond to the (0 0 2) plane, the (2 1 1) plane, the (1 1 2)plane, and the (2 0 2) plane for the hydroxyapatite mineral phase.Particularly suitable X-ray diffractometry peaks may be, for example, at26° and 31°, which correspond to the (0 0 2) plane, the (1 1 2) plane,and the (3 0 0) plane for carbonate-substituted hydroxyapatite. Othersuitable X-ray diffractometry peaks may be, for example, at 16°, 24°,and 33°, which correspond to the octacalcium phosphate mineral phase.Suitable spectra obtained by Fourier transform infrared spectrometryanalysis may be, for example, a peak at 450-600 cm⁻¹, which correspondsto O—P—O bending, and a peak at 900-1200 cm⁻¹, which corresponds toasymmetric P—O stretch of the PO₄ ³⁻ group of hydroxyapatite.Particularly suitable spectra peaks obtained by Fourier transforminfrared spectrometry analysis may be, for example, peaks at 876 cm⁻¹,1427 cm⁻¹, and 1483 cm⁻¹, which correspond to the carbonate (CO₃ ²)group. The peak for HPO₄ ²⁻ may be influenced by adjusting the calciumand phosphate ion concentrations of the SBF used to prepare the mineralcoating. For example, the HPO₄ ²⁻ peak may be increased by increasingthe calcium and phosphate concentrations of the SBF. Alternatively, theHPO₄ ²⁻ peak may be decreased by decreasing the calcium and phosphateconcentrations of the SBF. Another suitable peak obtained by Fouriertransform infrared spectrometry analysis may be, for example, a peakobtained for the octacalcium phosphate mineral phase at 1075 cm⁻¹, whichmay be influenced by adjusting the calcium and phosphate ionconcentrations in the simulated body fluid used to prepare the mineralcoating. For example, the 1075 cm⁻¹ peak may be made more distinct byincreasing the calcium and phosphate ion concentrations in the simulatedbody fluid used to prepare the mineral coating. Alternatively, the 1075cm⁻¹ peak may be made less distinct by decreasing the calcium andphosphate ion concentrations in the simulated body fluid used to preparethe mineral coating.

Energy dispersive X-ray spectroscopy analysis may also be used todetermine the calcium/phosphate ratio of the mineral coating. Forexample, the calcium/phosphate ratio may be increased by decreasing thecalcium and phosphate ion concentrations in the SBF. Alternatively, thecalcium/phosphate ratio may be decreased by increasing the calcium andphosphate ion concentrations in the SBF. Analysis of the mineralcoatings by energy dispersive X-ray spectroscopy allows for determiningthe level of carbonate (CO₃ ²) substitution for PO₄ ³⁻ and incorporationof HPO₄ ²⁻ into the mineral coatings. Typically, the SBF includescalcium and phosphate ions in a ratio of from about 10:1 to about 0.2:1,including from about 2.5:1 to about 1:1.

Further, the morphology of the mineral coatings may be analyzed byscanning electron microscopy, for example. Scanning electron microscopymay be used to visualize the morphology of the resulting mineralcoatings. The morphology of the resulting mineral coatings may be, forexample, a spherulitic microstructure, plate-like microstructure, and/ora net-like microstructure. Suitable average diameters of the spherulitesof a spherulitic microstructure may be, for example, from about 2 μm toabout 42 μm. Particularly suitable average diameters of the spherulitesof a spherulitic microstructure may be, for example, from about 2 μm toabout 4 μm. In another embodiment, particularly suitable averagediameters of the spherulites of a spherulitic microstructure may be, forexample, from about 2.5 μm to about 4.5 μm. In another embodiment,particularly suitable average diameters of the spherulites of aspherulitic microstructure may be, for example, from about 16 μm toabout 42 μm.

The method further includes contacting a polynucleotide with the mineralcoating. The polynucleotide may be contacted with the mineral coatingusing any method known in the art. For example, a solution of thepolynucleotide may be pipetted, poured, or sprayed onto the mineralcoating. Alternatively the mineral coating may be dipped in apolynucleotide solution. The polynucleotide binds to the mineral coatingby an electrostatic interaction between the polynucleotide and themineral coating.

Any polynucleotide may be contacted with the mineral coating for use inthe method of non-viral transfection. Suitable polynucleotides may be,for example, plasmids, oligonucleotides, small interfering RNAs(siRNAs), messenger RNA (mRNA), short hairpin RNAs (shRNAs), DNAaptamers, and RNA aptamers.

The polynucleotides may encode any protein of interest. For example, thepolynucleotides may encode proteins including growth factors andreporters. Particularly suitable proteins may be, for example, proteinsinvolved in the growth and the repair of bone such as, for example,BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, EGF, PDGFA, PDGFB,PDGFC, PDGFD, PDGFAB, VEGF-A, placenta growth factor (PIGF), VEGF-B,VEGF-C, VEGF-D, TGF-β1, TGF-β2, TGF-β3, AMH, ARTN, GDF1, GDF2, GDF3,GDF3A, GDF5, GDF6, GDF7, GDF8, GDF9, GDF10, GDF11, GDF15, GDFN, INHA,INHBA, INHBB, INHBC, INHBE, LEFTY1, LEFTY2, MSTN, NODAL, NRTN, PSPN,FGF1, FGF2, FGF3, FGF4, CBFA1/RUNX2, OSTERIZ, and OX9. Suitablereporters may be, for example, green fluorescent protein,chloramphenicol acetyl-trasferase, β-galactosidase, β-glucuronidase, andluciferase.

The method further includes contacting a cell with the mineral coatinghaving a polynucleotide and culturing the cell. Contacting the cellincludes seeding the cell to be transfected on the mineral coatinghaving the polynucleotide. The cells are contacted with the mineralcoating by adding a solution containing cells to the mineral coating andallowing cells in the solution to settle onto the mineral coating. Cellsmay be seeded, for example, at a density of from about 2.4×10⁴ cells/cm²to about 7.2×10⁴ cells/cm².

As the cells contact the mineral coating, the cells may change shape andform adhesions with the mineral coating. The cells may also migrate onthe mineral layer. Those skilled in the art will appreciate that, likeany cell culture system, the non-viral mineral coating is not a staticsystem.

After seeding, the cells are cultured on the mineral layer for anysuitable period of time. The suitable period of time may be for a periodof time such that the cells reach a particular confluence. For example,a suitable confluence may be from about 20% confluence to about 100%confluence. A particularly suitable confluence may be about 30%confluence. Those skilled in the art can determine the suitable amountof confluence for the particular cell type by determining the desiredpercent confluence at various percent confluence while culturing thecells up to about 100% confluence.

The method may include culturing the cells on the mineral coating havingthe polynucleotide with a polynucleotide release medium typicallyincluding calcium ions, phosphate ions, or a combination thereof.Culturing the cells in the polynucleotide release medium may allow forthe sustained release of the polynucleotide from the mineral coating andmake it available for transfection of the cell. The polynucleotiderelease medium may also cause the dissolution of the mineral layer suchthat calcium and phosphate ions are released from the mineral coatingand made available for transfection of the cell.

Suitable ion concentration of the polynucleotide release medium may befrom about 0 mM calcium chloride to about 2.5 mM calcium chloride andfrom about 0 mM potassium phosphate to about 1 mM potassium phosphate.Suitable pH of the polynucleotide-release medium may be, for example,from about 6.2 to about 7.8, including from about 7.2 to about 7.4.

Particularly suitable polynucleotide-release media may have, forexample, from about 0 mM calcium chloride to about 1.5 mM calciumchloride, from about 0 mM potassium phosphate to about 0.6 mM potassiumphosphate, and a pH of from about 4 to about 6.5. Without being bound bytheory, calcium and phosphate ions may influence polynucleotide uptakeand endosomal escape, leading to enhanced non-viral transfection. Thecalcium and phosphate ions may form calcium-phosphate nanoparticles nearthe dissolving mineral coating, which may enhance non-viral transfectionof the cells cultured on the mineral coatings.

High Throughput Platforms for Non-Viral Transfection

In another embodiment, the present disclosure is directed to a highthroughput platform for non-viral transfection. The high throughputplatform includes a substrate having a plurality of mineral coatings anda polynucleotide. Suitable substrates may be any type of plate or dishwith more than one well or chamber that allows for the comparisonbetween the wells or chambers. Particularly suitable substrates may be,for example, multiwell plates, multi-chambered plates, multiwell dishes,multiwell slides, and multiwell arrays such as those having, forexample, 6-, 12-, 24-, 48-, 96-, or more wells, chambers or arrays.

The substrates include the substrates previously described and may bemanufactured from a variety of materials. Suitable materials may be, forexample, polystyrene, polypropylene, polycarbonate, cycloolefins, glass,and combinations thereof.

A plurality of mineral coatings as described herein may be preparedaccording to the number of wells, chambers, and arrays contained on thesubstrates.

A polynucleotide is then contacted with the mineral coatings to bind thepolynucleotide to the mineral coating, as described herein. The amountof polynucleotide bound to the mineral coating may be controlled bycontrolling the morphology of the mineral coating. For example,polynucleotide binding to the mineral coating may be increased bydecreasing the calcium and phosphate in the SBF used to prepare themineral coating. Alternatively, polynucleotide binding to the mineralcoating may be decreased by increasing the calcium and phosphate in theSBF used to prepare the mineral coating.

After the polynucleotide is bound to the mineral coatings, cells may beseeded on the mineral coating, as described herein. Any suitable celltype may be cultured on the mineral coatings. Particularly suitable celltypes may be, for example, pluripotent stem cells, mesenchymal stemcells, and umbilical vein endothelial cells.

Cells may be cultured on the mineral coatings for any desired period oftime. Suitable time may be, for example, from less than 1 day to about 3weeks. Suitable times may be determined by those skilled in the artusing well known methods. For example, cells may be cultured for a timesufficient to reach a desired confluence. Cells may be cultured for atime sufficient to reach a desired level of transfection as determinedby detecting the expression level of the polynucleotide, detecting thepresence or absence of a marker, and conducting an enzyme assay, forexample.

Methods of Screening Non-Viral Transfection

In another aspect, the present disclosure is directed to a method ofscreening non-viral transfection of cells. The method includes culturinga plurality of cells on a plurality of mineral coatings comprising apolynucleotide bound to the mineral coatings, wherein the mineralcoatings are formed by hydrolyzing poly(α-hydroxy ester) coatings on asubstrate comprising a plurality of wells; incubating the hydrolyzedpoly(α-hydroxy ester) coatings in a plurality of simulated body fluidscomprising a calcium ion concentration of from about 5 mM to about 12.5mM, a phosphate ion concentration of from about 2 mM to about 12.5 mM, acarbonate ion concentration of from about 4 mM to about 100 mM, acalcium to phosphate ratio of from about 2.5:1 to about 1:1, and a pH offrom about 4 to about 7.5.

The method may further include analyzing transfection of the cells.Transfection may be analyzed using methods known by those skilled in theart. Suitable methods may be, for example, luminescence, fluorescence,enzyme linked immunosorbent assay (ELISA), Western blot analysis,Northern blot analysis, Southern blot analysis, polymerase chainreaction, and microscopy.

The disclosure will be more fully understood upon consideration of thefollowing non-limiting Examples.

EXAMPLES Example 1

Preparation of Mineralized Coatings on PLG Film

In this Example, mineral coatings were prepared on poly lactideglycolide (PLG) film.

Specifically, poly(lactide-co-glycolide) (“PLG”) (lactide:glycolide85:15, average molecular weight 50,000-70,000) was purchased fromSigma-Aldrich (St. Louis, Mo.). PLG was dissolved in chloroform anddried to make 1 cm×1 cm PLG films. PLG film was surface hydrolyzed in 1MNaOH solution for 1 hour to create a surface having COOH and OH groupsand rinsed in deionized (DI) water (18 MΩ cm). Various mineral solutionswere prepared by dissolving NaCl, KCl, MgSO₄, MgCl₂, NaHCO₃, CaCl₂,KH₂PO₄ and Tris base in DI water and adjusting the pH by adding HCl andNaOH for the supersaturated mineral solutions. Ion concentrations and pHvalues of the various mineral solutions are listed in Table 1 below.Mineral coatings were formed on PLG films by incubating the PLG films inthe various mineral solutions at 37° C. for 10 days. The mineralsolutions were refreshed daily and the resulting mineral-coated PLGfilms were rinsed in DI water and freeze dried until use.

TABLE 1 Ion Concentrations and pH Values of Mineral SolutionsConcentration (mM) Na⁺ K⁺ Ca²⁺ Mg²⁺ Cl⁻ HCO₃ ⁻ HPO₄ ²⁻ SO₄ ²⁻ pH Blood142.0 5.0 2.5 1.5 103.0 27.0 1.0 0.5 7.4 Plasma 2X 145.2 6.0 5.0 1.5 1574.2 2.0 0.5 6.8 SBF 3.5X 145.2 7.5 8.75 1.5 164.5 4.2 3.5 0.5 6.3 SBF 5X145.2 9.0 12.5 1.5 172 4.2 5.0 0.5 6.0 SBF

Example 2

Characterization of Mineral Coatings on PLG Films

In this Example, the mineral coatings on PLG films were characterized.

Specifically, the morphology of mineral coatings on PLG films wasexamined by scanning electron microscopy (SEM) (Carl Zeiss STM modelLEO-1530) operating at 5 keV. Mineral coated PLG films were sputtercoated with gold using a sputter coater (Denton Vacuum model DESK II)under 50 mTorr pressure, 40 mA current and a 35-second coating time. Tocharacterize the mineral composition, energy dispersive X-rayspectroscopy (EDS) analysis was performed in conjunction with SEM. AFourier transform infrared (FTIR) spectrometer (Bruker model EQUINOX 55)was used for further compositional analysis. Mineral coatings werescraped from the PLG films, mixed with potassium bromide, pressed into apellet and analyzed. All FTIR spectra were recorded in the range of400-2000 μm⁻¹. Hydroxyapatite powders (Sigma-Aldrich, St. Louis, Mo.)were used as a reference material. The crystal phases of mineralcoatings on PLG films were analyzed using X-ray diffractometry (XRD)(Broker AXS model HI-STAR). The mineral coatings scraped from PLG filmswere mounted using glass number 50 capillary tubes (Hampton Research,Aliso Viejo, Calif.) and analyzed under Cu Kα radiation. XRD spectrawere taken for 10 minute scanning in the range 2Θ=10-50°.

The morphology of mineral coatings formed on PLG films was influenced bythe calcium and phosphate ion concentrations, as well as pH of thesimulated body fluid. As shown in FIGS. 1A-1F, mineral coatings in allsimulated body fluid conditions had a spherulitic microstructure andplate-like nanostructure. The average diameter of mineral spherulitesformed in 2×SBF (3.1±0.92 μm) and 3.5×SBF (3.5±0.9 μm) weresignificantly smaller than mineral spherulites formed in 5×SBF(28.9±12.37 μm). The calcium and phosphate ion concentrations and the pHin SBF solution influenced both the phase (see, FIG. 2) and thecomposition (see, FIG. 3) of the mineral coatings. The appearance of twomain X-ray diffraction peaks at 26° and 31° indicated that the mineralcoatings were primarily composed of a hydroxyapatite mineral phase. The26° peak corresponds to the (0 0 2) plane of hydroxyapatite and the 31°peak corresponds to the (2 1 1), (1 1 2) and (2 0 2) planes.Carbonate-substituted hydroxyapatite has also been shown to displaypeaks at 26° and 31°, which correspond to the (0 0 2) plane and the (1 12) and (3 0 0) planes of carbonate-substituted hydroxyapatite. Thus, theXRD spectra were consistent with carbonate substitution in thehydroxyapatite mineral. Spectra from FTIR analysis showed two dominantpeaks that could be attributed to O—P—O bending and asymmetric P—Ostretch of the PO₄ ³⁻ group of hydroxyapatite (see FIG. 3). Vibrationpeaks at 876, 1427 and 1483 cm⁻¹, which could be assigned to thecarbonate (CO₃ ²⁻) group, were more strongly detected in mineralcoatings formed in 2×SBF when compared with those in 3.5× and 5×SBF.Thus, this result indicated that carbonate incorporation into mineralcoatings was enhanced in 2×SBF.

High calcium and phosphate ion concentrations and low pH of SBFsolutions provided favorable conditions for growth of an octacalciumphosphate (OCP) mineral phase. XRD spectra showed peaks at 16°, 24° and33°, which could be attributed to the OCP phase (see, FIG. 2). OCP peakswere more distinct when calcium and phosphate ion concentrations in SBFwere increased. Significantly, the lower initial pH of 3.5×SBF (pH 6.3)and 5×SBF (pH 6.0) may have provided a more favorable environment forOCP nucleation, which resulted in enhanced OCP content in the mineralcoatings. OCP is a less stable phase than hydroxyapatite underphysiological conditions.

Ca/P ratios measured by EDS analysis confirmed the presence of carbonateand HPO₄ ²⁻ ions measured by FTIR spectroscopy. EDS analysis indicatedthat the Ca/P ratio decreased with increasing calcium and phosphate ionconcentrations in SBF. Specifically, the Ca/P ratios of mineral coatingsproduced from 2×SBF (1.67±0.03) were higher than mineral coatingsproduced from 3.5×SBF (1.58±0.05) and 5×SBF (1.43±0.02). Thus, the Ca/Pratio decreased with increasing calcium and phosphate ion concentrationsdue to a decrease in CO₃ ²⁻ for PO₄ ³⁻ and increased incorporation ofHPO₄ ²⁻ at low pH.

Example 3

Effect of Solution Ion Concentrations and pH on Mineral Dissolution

In this Example, the effect of calcium and phosphate ion concentrationsand pH of SBF on mineral dissolution was examined

Specifically, mineral coated films were prepared as described above.Mineral coated films were incubated in DI water with 0.05 M Pipes bufferat pH 7.4 with varying amounts of CaCl₂ (0, 0.75, 1.5 or 2.5 mM) andKH₂PO₄ (0, 0.3, 0.6 or 1 mM). To determine the influence of pH onmineral dissolution, mineral coated films were incubated in DI waterwith 0.05 M Pipes buffer with 2.5 mM CaCl₂ and 1 mM KH₂PO₄ at pH 6.2,6.6, 7 and 7.4. Mineral coated films in 24-well plates were incubated in1 ml of each solution at 37° C. for 3 weeks. At specified times, eachsolution was assayed for soluble calcium and replaced with 1 ml of freshsolution. Mineral dissolution was determined by measuring calciumrelease into solution. To assay for soluble calcium, a 5 μl aliquot ofeach solution was added to a 195 μl calcium assay solution having 0.4 mMarsenazo III (MP Biomedicals, Solon, Ohio) in 0.02 M Tris base at pH7.4. Absorbance at 650 nm was converted to calcium concentration usingstandard curves relating absorbance intensity to calcium concentration.

Mineral dissolution increased over time in solutions with decreasingcalcium and phosphate ion concentrations at pH 7.4 (see, FIG. 4). Themineral dissolution rate was highest when the mineral coatings wereimmersed in a solution without calcium and phosphate ions (see, FIG. 5).Mineral dissolution decreased with increasing calcium and phosphate ionconcentrations in the dissolution environment, indicating that solutioncalcium ions were incorporated into the mineral coating during mineralreprecipitation. Calcium ion release from mineral coatings formed in2×SBF was less than calcium release from mineral coatings formed in3.5×SBF and 5×SBF in all dissolution environments tested. These resultssuggested that differences in dissolution rates may correspond todifferences in mineral morphology and composition, as well as theincreased presence of an OCP phase.

The total calcium release after 24 hours incubation increased linearlywith decreasing pH in the dissolution environments (see, FIG. 6).Calcium release from mineral coatings formed in 2×, 3.5× and 5×SBFsolutions reached maxima after 24 hours incubation at pH 6.2(0.150±0.214, 0.546±0.056 and 0.587±0.068 μmol, respectively). Theamount of calcium released into the surrounding environment graduallydecreased with increasing pH, indicating that calcium was being consumedduring mineral reprecipitation at higher pH (see, FIG. 7). Thedissolution rate of mineral coatings formed in 2×SBF was lower than thedissolution rates of mineral coatings formed in 3.5×SBF or 5×SBF in thepH range of 6.2-7.4. This result may be attributed to the increasedpresence of OCP phase in the minerals formed in 3.5×SBF and 5×SBF.

These results demonstrate that the ion concentration and pH of thesolution surrounding the mineral coating significantly influencedmineral stability.

Example 4

Plasmid Release from a Mineralized Substrate

In this Example, the release of plasmid DNA (“pDNA”) from a mineralizedsubstrate was examined

Specifically, pDsRed pDNA was amplified in competent TOF10F′ E. coli(Invitrogen, Carlsbad, Calif.) and purified using a Mega plasmidpurification kit (Qiagen, Valencia, Calif.). Mineral coated PLG filmswere incubated for 1 day in 1 ml of a pDNA buffer containing 20 mg pDNA(10 mM Tris-HCl, 1 mM EDTA, pH 7). Samples were rinsed with DI water andair dried. The amount of bound pDNA was calculated by subtracting theamount of pDNA that remained in the pDNA buffer from the initially addedamount of pDNA. The influence of calcium and phosphate ion concentrationon pDNA release was characterized by incubating samples in 0.05 Pipessolutions (pH 7.4) containing 0, 0.75, 1.5, or 2.5 M CaCl₂ and 0, 0.3,0.6, or 1 mM KH₂PO₄, respectively. The influence of pH on pDNA releasewas characterized by incubating samples in polynucleotide release mediacontaining 2.5 M CaCl₂ and 1 mM KH₂PO₄ and buffering these media witheither 0.05 M sodium acetate trihydrate (EMD, San Diego, Calif.) (pH 4or 5) or 0.05 M Pipes (pH 6 or 7.4). Each sample was incubated in 1 mlof polynucleotide release medium in 24-well plates at 37° C. for 3weeks. At specified times, polynucleotide release medium was removed foranalysis and replaced with 1 ml of fresh polynucleotide release medium.A 50 μl aliquot of the polynucleotide release medium was added to 150 μlof a working solution prepared using a Quant-iT Picogreen dsDNA assaykit (Invitrogen, Carlsbad, Calif.). The fluorescence measured at 520 nmwas converted to an amount of released pDNA using standard curvesprepared with known concentrations of pDNA in solution.

The amount of bound pDNA on the mineral coatings formed in 2×SBF(15.8±1.5 mg) was significantly higher than that on mineral coatingsformed in 3.5×SBF (6.1±1.3 mg) or 5×SBF (3.0±0.6 mg). The release ratesof pDNA increased with decreasing concentrations of calcium andphosphate ions in the polynucleotide release media, with the largestamount of pDNA released into a polynucleotide release medium devoid ofcalcium and phosphate ions.

The total amount of pDNA released over time significantly increased asthe pH of the polynucleotide release medium decreased. At nearphysiological pH, the release of pDNA was hindered. As shown in FIG. 9,pH-dependent release of pDNA was directly related to pH-dependentdissolution of mineral coatings. Acidic conditions destabilized themineral structure more quickly than near neutral media, resulting ingreater pDNA release in the low pH condition.

Example 5

Effect of Mineral Coating Morphology on Multipotent Stem Cell Behaviors

In this Example, the effect of mineral coating morphology on multipotentstem cell behaviors was examined

Specifically, mineral coatings were formed in 2×SBF, 3.5×SBF and 5×SBFsolutions containing different calcium and phosphate concentrations andCa/P ratios as shown in Table 2. As demonstrated in the Examplesdescribed above, the morphology of mineral coatings was modulated bychanging calcium and phosphate concentrations and Ca/P ratios withoutcreating significant differences in composition, crystallinity andsolubility.

TABLE 2 Ion concentrations for Ca/P ratio effect on mineral properties(mM). [Ca²⁺] [Ca²⁺] in blood plasma 2X SBF 3.5X SBF 5X SBF Ca/P 2.5 21.5 1 2.5 2 1.5 1 2.5 2 1.5 1 Ca²⁺ 5 5 5 5 8.8 8.8 8.8 8.8 12.5 12.512.5 12.5 PO₄ ³⁻ 2 2.5 3.3 5 3.5 4.4 5.8 8.8 5 6.3 8.3 12.5 CO₃ ²⁻ 4.24.2 4.2 4.2 4.2 4.2 4.2 4.2 4.2 4.2 4.2 4.2 pH 6.8 6.6 6.5 6.2 6.1 5.85.8 5.7 5.8 5.7 5.5 5.3

Multipotent stem cells (C₃H₁₀T½ and human mesenchymal stem cells(“hMSC”)) (about 30% of confluence on mineral surface) were seeded withDMEM 10% cosmic calf serum and MEM 10% fetal bovine serum and culturedfor 4 days and 8 days. At days 4 and 8, cells were lysed and thesupernatant was assayed using a cell proliferation kit (Invitrogen,Carlsbad, Calif.) to measure the total DNA amount. Additionally, hMSCproliferation on mineral coatings during culture in minimum essentialmedium (MEM) with 10% fetal bovine serum (FBS) and MEM containing 10%FBS with osteogenic supplement (OS) (50 μM ascorbic acid-2-phosphate, 10mM β-glycerophosphate, and 100 nM dexamethasone) was characterized todetermine the effect of OS on hMSC differentiation on mineral coatings.

As illustrated in FIG. 10, the cell density decreased when morphology ofmineral coatings was changed from spherulitic microstructure toplate-like or net-like structure. As shown in FIGS. 11A and 11B, cellproliferation on mineral coatings increased with increasing incubationtime in cell culture media. Additionally, hMSC proliferation on mineralcoatings in MEM+OS was decreased as compared to hMSC proliferation onmineral coatings in MEM.

Example 6

Control of Mineral Coating Dissolution in Cell Culture Conditions

In this Example, the effect of mineral morphology on mineralcrystallinity in cell culture conditions was examined

Specifically, PLG films were mineralized as described above using SBFsolutions containing different concentrations of calcium, phosphate, andcarbonate and mineral properties were analyzed. Additionally, Ca/P ratioand crystallinity index of mineral coatings with dissolution rate ofmineral coatings were compared to determine the change in mineralstability. The dissolution of mineral coatings in cell culture medium(serum-free DMEM) was determined by measuring the change of calciumamount over time.

As shown in FIGS. 13 and 14, the calcium/phosphate ratios andcrystallinity of mineral coatings grown in 2×SBF, 3.5×SBF, and 5×SBFhaving varying carbonate concentrations varied. Additionally, as shownin FIG. 15, mineral stability increased with mineral crystallinity.FIGS. 16A-16C show that the mineral coating dissolution can occur inDMEM serum-free medium and can be controlled by growing the mineralcoatings in specific SBF and varying the carbonate concentrations in theSBF. These results demonstrated that controlling mineral coatingmorphology with subtle change of mineral crystallinity can allow forcontrolling mineral dissolution in cell culture conditions.

Example 7

Control of Transfection by Modulating Mineral Stability

In this Example, the effect of mineral stability on transfection ofcells cultured on mineral coatings was examined.

Mineral coatings were prepared in 2×SBF, 3.5×SBF and 5×SBF havingvarying carbonate concentrations as described above. pDNA complexes wereimmobilized on the mineral coatings by adsorption and co-precipitationmethods. pDNA encoding secreted luciferase was used to allow for thecontinuous measurement of target protein without requiring cell lysis.For transfection, multipotent stem cells (C3H10T½ and hMSC) were seededat a density of 1×10⁴ cell per well with DMEM with 10% cosmic calf serum(C3H10T½) and MEM with 10% FBS (hMSC). To determine the transfectionrate of cells from dissolving mineral coatings, the luminescence ofsecreted luciferase after 2 days of cell seeding was screened using anin vivo image system. At specific time points, cell culture mediumcontaining secreted luciferase was collected and assayed with Cell-Glowluciferase kit (Clontech, Mountain View, Calif.). To measure luciferaseactivity, luminescence was measured using a microplate luminometer(Veritas, Sunnyvale, Calif.). Transfection of C3H10T½ and hMSC wasmeasured by dividing luciferase activity by cell viability, which wasmeasured using CellTiter-Blue cell viability assay kit (Promega,Madison, Wis.).

As shown in FIG. 17, pDNA complex binding was dependent on the bindingmethod. As shown in FIGS. 18A and 18B, luciferase activity ofC3H10T½(FIG. 18A) and hMSC (FIG. 18B) after 2 days culture on mineralcoatings indicated a significant difference compared to 2×−4.2 conditionby adsorption and co-precipitation. The transfection of cells on mineralcoatings containing adsorbed pDNA complexes was correlated with themineral stability change. These experiments demonstrated that non-viraltransfection of cells can be controlled by changing mineral properties.These results also demonstrated that the relative luminescent intensitycan be screened in an enhanced high throughput format using an in vivoimage system. These results further demonstrated that many differentmineral coatings can be prepared that can then be used as a platform forhigh throughput transfection of cells for screening.

Example 8

Transfection of Human Umbilical Vein Endothelial Cells on MineralCoatings

In this Example, transfection of human umbilical vein endothelial(“HUVEC”) cells cultured on mineral coatings was examined

Specifically, HUVEC were cultured on mineral coatings in M199 mediumwith 20% FBS and EGM-2 after immobilization of pDNA complexes encodingsecreted luciferase on mineral coatings by adsorption andco-precipitation methods. At specific time points, cell culture mediacontaining secreted luciferase were collected for luciferase assay asdescribed above. Cell viability was measured using a microplate reader(Bio Tek, Winooski, Vt.). The results are shown FIG. 19.

As shown in FIG. 19, combinatorial changes in mineral properties andpDNA complex amount identified conditions in which the luciferaseactivity per cell was increased by 11-fold for primary HUVECs. This foldincrease is relative to standard manufacturer protocols usingpDNA-Lipofectamine complexes in solution. In addition to the high levelsof transfection relative to standard commercial techniques,combinatorial variation in mineral properties and pDNA complex amountsalso resulted in controllable levels of transfection, and collectivelythe data show conditions in which the luciferase activity can be variedover a broad range of values.

Example 9

In this Example, non-viral human mesencymal stem cell(hMSC)-transfection using mineral coated 3D scaffold array was examined.

PLG scaffolds were fabricated in a poly(propylene) 96-well plate usingPLG (10% w/v) in acetone with a salt fusion/solvent casting/saltleaching technique. NaCl particles were previously sieved to 250-425 μmand used as porogen. 130 mg NaCl was placed in each well of the 96-wellplate and incubated in a 95% humidity cell culture incubator at 37° C.for 4 h for salt fusion. Then the fused salt template was driedovernight in an oven at 50° C. 30 μL of the PLG solution was added intoeach well and the whole plate was centrifuged at 2000 RPM to wet all theNaCl particles. After evaporating the solvent, the whole plate was thenimmersed in a 4.0 L beaker filled with DI water to leach out the saltparticles. The water was refreshed every 4 h and the leaching processtook approximately 48 h.

To coat the PLG scaffolds with mineral, the scaffold was treated with200 μL 0.1 M NaOH for 5 min at room temperature to activate the COOH andOH groups on the polymer surface. 200 μL of various SBF solutions (Table3) was then added into each well of scaffolds after extensively washingout the NaOH residue. The SBF solution was renewed every 12 h tomaintain a consistent ionic strength for 7 days. The morphology of thePLG scaffold before and after mineral coatings was observed usingscanning electron microscopy. Mineral coated PLG scaffold was stainedusing Alizarin Red S (Sigma-Aldrich, St. Louis, Mo.) for identifyingcalcium on the scaffold.

TABLE 3 Ion concentrations for CO₃ ²⁻ effect on mineral properties(unit: mM) [Ca²⁺] & [PO₄ ³⁻] [Ca²⁺] & [PO₄ ³⁻] in blood plasma 2X 3.5X5X Ca²⁺ 5 5 5 5 8.8 8.8 8.8 8.8 12.5 12.5 12.5 12.5 PO₄ ³⁻ 2 2 2 2 3.53.5 3.5 3.5 5 5 5 5 CO₃ ²⁻ 4.2 25 50 100 4.2 25 50 100 4.2 25 50 100 pH6.8 6.8 6.8 6.8 6.1 6.1 6.1 6.1 5.8 5.8 5.8 5.8

The gross view of the 3D PLG scaffold is shown in FIG. 20A. Highlyporous scaffold was generated using the current approach. The morphologyof the 3D scaffold was also examined by scanning electron microscopy andshown in FIG. 20C. The pore size of the scaffold was approximatelybetween 250-400 μm. The mineral coating formed on the scaffold wasillustrated by Alizarin Red S staining which showed uniform mineralcoating formed through the whole scaffold (FIG. 20B). The morphology ofthe coating was also visualized by electron microscopy which also showedthe coverage of the mineral over the scaffold structure while retainingthe original porous structure (FIG. 20D).

The procedure for the transfection was similar to that of mineralcoatings on PLG surfaces with some adjustments: 1 μg plasmid DNA wasused for each scaffold; plasmid DNA binding was conducted in 100 μLpDNA/Lipofectamine 2000 complex containing medium; hMSC was seeded onthe mineral coated scaffolds with complexes at a density of 1.0×10⁵cells/scaffold. After 2 d of cell culture on mineral scaffolds, eachmedium was taken for luciferase activity assay and total protein amounton each scaffold was measured by micro BCA kit (Thermo Scientific,Rockford, Ill.).

This Example exhibits that cell transfection could also be achieved bymineral coating in a 3D scaffold format. The luciferase activity ofcoating with high carbonate content generally showed higher celltransfection efficiency. The peak value of transfection was observedwhen 2×−50 SBF was used to form the mineral coating (FIG. 21).

The examples described above demonstrate that the mineral coatings andtransfection methods offer the ability to non-virally transfect cells.Surprisingly, use of the mineral coatings of the present disclosure fortransfection showed 150-fold greater transfection rates when compared toother commercially-available transfection methods. The mineral coatingsand transfection methods of the present disclosure further allow for ahigh throughput systematic screen for controlling and enhancing thetransfection of cells.

In view of the above, it will be seen that the several advantages of thedisclosure are achieved and other advantageous results attained. Asvarious changes could be made in the above methods and systems withoutdeparting from the scope of the disclosure, it is intended that allmatter contained in the above description and shown in the accompanyingdrawings shall be interpreted as illustrative and not in a limitingsense.

When introducing elements of the present disclosure or the variousversions, embodiment(s) or aspects thereof, the articles “a”, “an”,“the” and “said” are intended to mean that there are one or more of theelements. The terms “comprising”, “including” and “having” are intendedto be inclusive and mean that there may be additional elements otherthan the listed elements.

What is claimed is:
 1. A method of non-viral transfection comprising:preparing a substrate comprising a mineral coating, wherein the mineralcoating is formed by incubating the substrate in a simulated body fluid,wherein the simulated body fluid comprises from about 5 mM to about 12.5mM calcium ions, from about 2 mM to about 12.5 mM phosphate ions, fromabout 4 mM to about 100 mM carbonate ions, and a pH of from about 4 toabout 7.5; contacting the substrate comprising the mineral coating witha polynucleotide-lipid complex, wherein the polynucleotide-lipid complexadsorbs to the mineral coating; contacting a cell with the mineralcoating, wherein the cell is transfected with the polynucleotide; andculturing the cell.
 2. The method of claim 1, wherein the mineralcoating comprises a calcium to phosphate ratio of from about 2.5:1 toabout 1:1.
 3. The method of claim 1, wherein the substrate furthercomprises a poly(α-hydroxy ester).
 4. The method of claim 3, wherein thepoly(α-hydroxy ester) is selected from the group consisting ofpoly(L-lactide), poly(lactide-co-glycolide), poly(ϵ-caprolactone), andcombinations thereof.
 5. The method of claim 1, further comprisingculturing the cell in a polynucleotide release medium, wherein thepolynucleotide release medium comprises an ion selected from the groupconsisting of calcium, phosphate, and combinations thereof, wherein thecalcium ion concentration comprises from about 0 mM to about 2.5 mM, andthe phosphate ion concentration comprises from about 0 mM to about 1 mM.6. The method of claim 1, further comprising culturing the cell in apolynucleotide release medium, wherein the polynucleotide release mediumcomprises a pH of from about 6.2 to about 7.8.
 7. The method of claim 1,wherein the mineral coating comprises a morphology selected from thegroup consisting of a spherulitic microstructure, a plate-likemicrostructure, a net-like microstructure, and combinations thereof. 8.The method of claim 7, wherein the spherulitic microstructure comprisesan average spherulite diameter of from about 2 μm to about 4 μm.
 9. Themethod of claim 1, wherein the cell is selected from the groupconsisting of a pluripotent stem cell, a mesenchymal stem cell, and anumbilical vein endothelial cell.
 10. The method of claim 1, wherein thepolynucleotide is contacted with the mineral coated substrate by amethod selected from pipetting, pouring, and spraying the polynucleotideonto the mineral coated substrate.